Oligonucleotide chemistry is central to the advancement of core technologies such as DNA sequencing, forensic and genetic analysis and has impacted greatly on the discipline of molecular biology. Oligonucleotides and their analogues are essential tools in these areas. They are often produced by automated solid-phase phosphoramidite synthesis. However, this process can only assemble DNA strands up to about 150 bases in length. Synthesis of long RNA strands is more difficult owing to problems caused by the presence of the 2′-hydroxyl group of ribose which requires selective protection during oligonucleotide assembly. This reduces the coupling efficiency of RNA phosphoramidite monomers due to steric hindrance. In addition, side-reactions which occur during the removal (or premature loss) of the 2′-protecting groups cause phosphodiester backbone cleavage and 3′ to 2′ phosphate migration. Although several ingenious strategies have been developed to minimise these problems and to improve the synthesis of long RNA molecules, the chemical complexity of solid-phase RNA synthesis dictates that constructs longer than 50 nucleotides in length remain difficult to prepare. Most biologically important DNA and RNA molecules for example genes, ribozymes, aptamers and riboswitches are significantly longer than the length that is currently achievable by solid-phase synthesis, so new approaches to the synthesis of long DNA and RNA molecules are urgently required.
Although DNA and RNA synthesis by enzymatic replication or transcription might seem a viable alternative, it does not permit the site-specific incorporation of multiple modifications at sugars, bases, or phosphates and also leads to the loss of epigenetic information such as DNA methylation.
In contrast, automated solid-phase DNA and RNA synthesis is compatible with the introduction of methylated nucleotides, fluorescent tags, isotopic labels (for NMR studies) and other groups to improve biological activity and resistance to enzymatic degradation. The scope and utility of important DNA and RNA constructs can be significantly extended by such chemical modifications.
Another drawback of enzymatic replication or transcription is that the DNA and RNA products can only be cost-effectively produced at a small scale. The scale of chemical synthesis, by contrast, is potentially unlimited.
Previous studies have attempted to chemically ligate synthesized oligonucleotides to form longer DNA molecules as described in WO2008/120016, Kumar et al. 2007, J Am Chem Soc 129, 6859-6864, Kocalka et al. 2008, Chem Bio Chem, 9, 1280-1285, and El-Sagheer et al. 2009, J Am Chem. Soc. 131(11), 3958-3964. The drawback with these molecules was that, because they contained unnatural linkages between the oligonucleotides they were not fully active in a biological system. DNA and RNA polymerases could not read these nucleotide sequences accurately and mis-read or missed out nucleotides when trying to replicate the sequences.
Enzymatic ligation using, for example T4 DNA ligase can be used to join oligonucleotides but the use of ligases has other drawbacks; they are often contaminated with RNase enzymes which can partially degrade the ligation products, and the ligation protocols require subsequent removal of the ligase protein to produce pure DNA or RNA. Moreover, enzymatic ligation methods are not suitable for the large scale synthesis of DNA or RNA, and the yields of enzymatic ligation are sometimes low, particularly when using chemically modified DNA or RNA substrates or mixed DNA/RNA strands.
It would therefore be advantageous to provide a method that can be used on an industrial scale and can synthesize long DNA and RNA molecules that can be read correctly by DNA and RNA polymerases and hence can be used for in vitro and in vivo applications including applications in biology and nanotechnology.